Compositions of Engineered Human Arginases and Methods for Treating Cancer

ABSTRACT

Compositions and methods for the treatment of cancer are described, and, more preferably, to the treatment of cancers that do not express, or are otherwise deficient in, trgininosuccinate synthetase, with enzymes that deplete L-Arginine in serum. In one embodiment, the present invention contemplates an arginase protein, such as a human Arginase I protein, comprising at least one amino acid substitution and a metal cofactor, said protein comprising an increased catalytic activity when compared with a native human Arginase I.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/275,259, filed May 12, 2014, which is a continuation of Ser.No. 13/863,448, filed Apr. 16, 2013, which is a continuation of U.S.patent application Ser. No. 12/610,685, filed Nov. 2, 2009, now U.S.Pat. No. 8,440,184, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/110,218, filed Oct. 31, 2008, the entiredisclosure of each are specifically incorporated herein by reference.

The sequence listing that is contained in the file named“GGEOP0002USC2_ST25.txt”, which was created on Nov. 17, 2014, is filedherewith by electronic submission and is incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to compositions and methods for thetreatment of cancer with enzymes that deplete L-Arginine in serum. Insome embodiments, the cancer is one that does not express, or isotherwise deficient in, argininosuccinate synthetase (ASS), ornithinetranscarbamylase (OTC), or other enzymes required for argininebiosynthesis.

2. Description of the Related Art

It has been recognized for over 50 years that certain tumor cells have ahigh demand for amino acids, such as L-Arginine and are killed underconditions of L-Arginine depletion (Wheatley and Campbell, 2002). Inhuman cells L-Arginine is synthesized in two steps; firstargininosuccinate synthetase (ASS) converts L-Citrulline and aspartateto argininosuccinate, followed by conversion of argininosuccinate toL-Arginine and fumarate by argininosuccinate lyase. L-Citrulline itselfis synthesized from L-Omithine and carbamoyl phosphate by the enzymeornithine transcarbamylase (OTC). A large number of hepatocellularcarcinomas, melanomas and, as discovered recently, renal cell carcinomas(Ensor et al., 2002; Feun et al., 2007; Yoon et al., 2007) do notexpress ASS and thus are sensitive to L-Arginine depletion. Themolecular basis for the lack of ASS expression appears to be diverse andincludes aberrant gene regulation and splicing defects. Whereasnon-malignant cells enter into quiescence (G_(o)) when depleted ofL-Arginine and thus remain viable for several weeks, tumor cells havecell cycle defects that lead to the re-initiation of DNA synthesis eventhough protein synthesis is inhibited, in turn resulting in majorimbalances and rapid cell death (Shen et al., 2006; Scott et al., 2000).The selective toxicity of L-Arginine depletion for HCC, melanoma andother ASS-deficient cancer cells has been extensively demonstrated invitro, in xenograft animal models and in clinical trials (Ensor et al.,2002; Feun et al., 2007; Shen et al., 2006; Izzo et al., 2004). RecentlyCheng et al. (2007) demonstrated that many HCC cells are also deficientin ornithine transcarbamylase expression and thus, they are alsosusceptible to enzymatic L-Arginine depletion.

There is interest in the use of L-Arginine hydrolytic enzymes for cancertherapy, especially the treatment of hepatocarcinomas, melanomas andrenal cell carcinomas, which are common forms of cancer associated withhigh morbidity. Two L-Arginine degrading enzymes have been used forcancer therapy: bacterial arginine deiminase and human Arginases.Unfortunately, both of these enzymes display significant shortcomingsthat present major impediments to clinical use (immunogenicity and lowcatalytic catalytic activity and very poor stability in serum,respectively). Thus, the therapeutic success of L-Arginine depletiontherapy will rely on addressing these shortcomings.

SUMMARY OF THE INVENTION

The invention generally relates to compositions and methods for thetreatment of cancer with enzymes that deplete L-Arginine in serum. Insome embodiments, the cancer is one that does not express, or isotherwise deficient in, argininosuccinate synthetase (ASS), ornithinetranscarbamylase (OTC), or other enzymes required for argininebiosynthesis.

In some aspects, the present invention contemplates arginase proteinswherein the natural metal cofactor (Mn²⁺) is replaced with anothermetal. In particular embodiments, the arginase protein comprises anamino acid sequence of human Arginase I or an amino acid sequence ofhuman Arginase II and a non-native metal cofactor. In some embodiments,the metal is cobalt (Co²⁺). Human Arginase I and II proteins of thepresent invention have two Mn(II) sites; either or both sites can besubstituted so as to generate a mutatated Arginase I or II protein witha non-native metal cofactor. In some embodiments, the protein displays ak_(cat)/K_(m) greater than 400 mM⁻¹ s⁻¹ at pH 7.4. In a particularembodiment, the protein displays a k_(cat)/K_(m) between 400 mM⁻¹ s⁻¹and 4,000 mM⁻¹ s⁻¹ at pH 7.4. In another embodiment, the proteindisplays a k_(cat)/K_(M) between 400 mM⁻¹ s⁻¹ and 2,500 mM⁻¹ s⁻¹ at pH7.4 at 37° C. In a particular embodiment, the present inventioncontemplates a protein comprising an amino acid sequence of humanArginase I or II and a non-native metal cofactor, wherein said proteinexhibits a k_(cat)/K_(m) greater than 400 mM⁻¹ s⁻¹ at 37° C., pH 7.4.

In some embodiments, the native arginase is modified only by thesubstitution of the metal cofactor. In other embodiments, the arginaseis modified by substitution of the metal cofactor in addition to othermodifications, such as substitutions, deletions, and truncations. In aparticular embodiment, the invention provides a protein comprising anative amino acid sequence of human Arginase I or II and a non-nativemetal cofactor, wherein the amino acid sequence is lacking part of thenative sequence. In particular embodiments, the non-native metalcofactor is cobalt. In some embodiments, the amino acid sequence ofhuman Arginase I comprises SEQ ID NO:13. In other embodiments, the aminoacid sequence of human Arginase II comprises SEQ ID NO:14. In yet otherembodiments, the arginase lacks a portion of the wild-type sequence. Inother embodiments, the amino acid sequence comprises a truncatedArginase I or Arginase II sequence. In a particular embodiment, thearginase is Arginase II and lacks the first 21 amino acids of thewild-type sequence. In another embodiment, the native arginases lacks anN-terminal methionine.

In another aspect, the present invention contemplates an arginaseprotein comprising at least one amino acid substitution, wherein theprotein displays an increased catalytic activity under physiologicalconditions and especially at the pH of human serum (pH 7.4) whencompared with native human Arginase I or II protein. In someembodiments, the arginase protein is a human Arginase I protein or humanArginase II protein. In some embodiments, the protein further comprisesa non-native metal cofactor. In particular embodiments, the non-nativemetal cofactor is Co⁺². Substitution of the Mn⁺² cofactor with Co⁺²results in marked increase in catalytic activity and a drastic reductionin K_(m) at physiological pH.

In one embodiment, the present invention provides a human Arginase Iprotein comprising at least one amino acid substitution at the metalbinding site, wherein the protein displays an increase in the hydrolysisof Arginine that results in a k_(cat)/K_(m) of at least two fold greaterthan that of a native human Arginase I having SEQ ID NO:13. In anotherembodiment, the present invention provides a human Arginase II proteincomprising at least one amino acid substitution at the metal bindingsite, wherein the protein displays an increase in the hydrolysis ofArginine that results in a k_(cat)/K_(m) of at least two fold greaterthan that of a native human Arginase II having SEQ ID NO:14. In someembodiments, the protein displays a k_(cat)/K_(m) greater than 400 mM⁻¹s⁻¹ at pH 7.4. In a particular embodiment, the protein displays ak_(cat/)K_(m) between 400 mM⁻¹ s⁻¹ and 4,000 mM⁻¹ s⁻¹ at pH 7.4. Inanother embodiment, the protein displays a k_(cat)/K_(m) between 400mM⁻¹ s⁻¹ and 2,500 mM⁻¹ s⁻¹ at pH 7.4 at 37° C. In some aspects, theinvention provides mutations that increase the stability of humanarginases in serum relative to the stability of native human arginases.

In some embodiments, the amino acid substitution is at His101, Asp124,His126, Asp128, Asp232, Asp234, Trp122, Asp181, Ser230, His120, Asp143,His145, Asp147, Asp251, Asp253, Trp141, Asp200, Ser249, Cys303, orGlu256. A number of mutations have been found to increase the catalyticactivity and drastically reduce the K_(m) for L-Arginine underphysiological conditions. In some embodiments, mutations aresubstitution mutations selected from the group consisting of Asp181Ser,Ser230Cys, Ser230Gly, Cys303Phe, Cys303Ile, Glu256Gln, Asp181Glu andSer230Ala. In some aspects, the present invention provides embodimentswhere two or more mutations are introduced in human arginase. In someembodiments, the human arginase protein comprises at least two aminoacid substitutions. In a particular embodiment, the substitutions areAsp181Glu and Ser230Ala.

In some aspects, the present invention provides arginases comprisingadditional changes relative to the wild-type or native protein. In someembodiments, the changes include substitution, deletions (e.g. lackingpart of the native sequence), truncations, or a combination thereof. Insome embodiments, the present invention also contemplates nativearginases, wherein the only amino acid sequence changes are deletions.In a particular embodiment, the present invention contemplates a humanArginase I protein, wherein the protein lacks an N-terminal methionine.Other and larger deletions are also contemplated for the various mutantarginases described herein. For example, truncated Arginase lacking the14 C-terminal amino acids has been reported, leaving Arg-308 as the lastresidue in the sequence (Mora et al., 2000). In yet another embodiment,the arginase lacks the first 21 amino acids of the wild-type sequence.

In some aspects, the present invention also contemplates fusion proteinscomprising an arginase linked to a non-arginase amino acid sequence. Inone embodiment, the non-arginase sequence comprises at least a portionof the Fc region of an immunoglobulin, e.g., to increase the half-lifeof the arginase in serum when administered to a patient. The Fc regionor portion thereof may be any suitable Fc region. In one embodiment, theFc region or portion thereof is an IgG Fc region. In some embodiments,the amino acid sequence having arginase activity is selected from thegroup consisting of a native or mutated amino acid sequence of humanArginase I and a native or mutated amino acid sequence of human ArginaseII. In one embodiment, a dimeric Fc-Arginase fusion protein iscontemplated.

The arginase in the fusion protein may be native, mutated, and/orotherwise modified, e.g., metal cofactor modified. In some embodiments,the arginase may contain deletions, substitutions, truncations or acombination thereof. In a particular embodiment, the present inventioncontemplates an Fc-arginase containing fusion protein, wherein thearginase is an Arginase I. In one embodiment, the arginase lacks aportion of the wild-type sequence. In another embodiment, the arginaseis Arginase I lacking an N-terminal methionine. In yet anotherembodiment, the arginase is Arginase II, wherein the Arginase II lacksthe first 21 amino acids of the wild-type Arginase II sequence. In someembodiments, the arginase further comprise a non-native metal cofactor.In these embodiments, either or both sites can be substituted togenerate a fusion protein comprising an amino acid sequence of humanArginase I or II and a non-native metal cofactor. In some embodiments,the non-native metal cofactor is cobalt. In some embodiments, thearginase contains a substitution. In one embodiment, the substitution isGlu256Gln. In another embodiment, the substitution is Asp181Ser. In yetanother embodiment, the substitution is Ser230Cys. In still anotherembodiment, the substitution is Ser230Gly. In yet another embodiment,the substitution is Cys303Phe. In still another embodiment, thesubstitution is Cys303Ile. In some embodiments, the human Arginase Icomprises at least two amino acid substitutions. In one embodiment, thesubstitutions are Asp181Glu and Ser230Asp.

In some aspects, the present invention further contemplates nucleic acidencoding such arginases. In some embodiments, the nucleic acid that hasbeen codon optimized for expression in bacteria. In particularembodiments, the bacteria is E. coli. In other aspects, the presentinvention further contemplates vectors containing such nucleic acids. Inparticular embodiments, the nucleic acid encoding the mutant arginase isoperably linked to a promoter, including but not limited to heterologouspromoters. In still further aspects, the present invention furthercontemplates host cells comprising such vectors. In some embodiments,the host cells are transfected or transformed host cells expressing themutant arginases. The proteins may be expressed in any suitable manner.In one embodiment, the proteins are expressed in a host cell such thatthe protein is glycosylated. In another embodiment, the proteins areexpressed in a host cell such that the protein is aglycosylated.

The present invention also contemplates methods of treatment by theadministration of the arginase proteins of the present invention, and inparticular methods of treating subjects with cancer. In someembodiments, the cancer is one that does not express, or is otherwisedeficient in, argininosuccinate synthetase (ASS) or ornithinetranscarbamylase (OTC). In particular embodiments, the human cancer isan arginine auxotrophic cancer. As discussed above, the arginase proteinmay be native, mutated, and/or otherwise modified, e.g., metal cofactormodified. In one embodiment, the present invention contemplates a methodof treating a human cancer patient comprising administering aformulation comprising a fusion protein, the fusion protein comprisingan amino acid sequence having arginase activity and at least a portionof the Fc region of a human immunoglobulin to the patient. In someembodiments, the administration occurs under conditions such that atleast a portion of the cancer cells of the cancer are killed. In anotherembodiment, the formulation comprises an amino acid sequence havinghuman arginase activity higher than that displayed by the authentichuman arginases at physiological conditions and further comprising anattached polyethylene glycol chain. In some embodiment, the formulationis a pharmaceutical formulation comprising any of the above discussedarginase proteins and a pharmaceutically acceptable excipients. Suchpharmaceutically acceptable excipients are well known to those havingskill in the art. All of the above arginase variants are contemplated asuseful for human therapy.

The cancer may be any type of cancer or tumor type. In some embodiments,the cancer is hepatocellular carcinoma, renal cell carcinoma, melanoma,prostate cancer, or pancreatic cancer. In some embodiments, theformulation is administered topically, intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intraocularly, intranasally, intravitreally, intravaginally,intrarectally, intramuscularly, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,orally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, or via a lavage. In one embodiment, to increase serumhalf-life, the arginase variants described herein are “pegylated.”

All of the above mentioned arginases, variants and the like arecontemplated in a preferred embodiment as purified or isolated proteins,and preferably monomeric proteins.

The embodiments in the Example section are understood to be embodimentsof the invention that are applicable to all aspects of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

The term “therapeutically effective” as used herein refers to an amountof cells and/or therapeutic composition (such as a therapeuticpolynucleotide and/or therapeutic polypeptide) that is employed inmethods of the present invention to achieve a therapeutic effect, suchas wherein at least one symptom of a condition being treated is at leastameliorated, and/or to the analysis of the processes or materials usedin conjunction with these cells.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows Arginase I (SEQ ID NO:1) and Arginase II (SEQ ID NO:2)nucleic acid sequences.

FIG. 2 is a photograph of a SDS-PAGE showing purification steps forhuman Arginase I. L=Molecular Weight Ladder; WC=Whole Cell Fraction,SN=Supernatant; FT=Flow Through from IMAC Column; W=Column Wash;E=Arginase Elution Fraction.

FIG. 3 is a representative graph of steady-state kinetics of L-argininehydrolysis by Co-hArgI () and Mn-hArgI (∘) in a 100 mM Hepes buffer, pH7.4, 37° C. Co-hArgI had a k_(cat) of 240±14 s⁻¹, a K_(M) of 190±40 μM,and k_(cat)/K_(M) of 1,270±330 mM⁻¹ s⁻¹. Mn-Argl had a k_(cat) of 300±12s⁻¹, a K_(M) of 2,330±260 uM, and k_(cat)/K_(M) of 129±20 mM⁻¹ s⁻¹.

FIG. 4 is a plot of k_(cat)/K_(M) versus pH for Co-hArgI () with anascending limb pK_(a) of 7.5 and Mn-hArgI (▪) with an ascending limbpK_(a) of 8.5.

FIG. 5 is a graph showing the stability of Co-hArgI and Mn-hArgI (1 μM)incubated in pooled human serum at 37° C. over time in pooled humanserum. Aliquots were withdrawn over time and assayed against 1 mM ofL-Arg in a 100 mM Hepes buffer, pH 7.4, at 37° C. Mn-hArgI (∘) displayedan exponential loss of activity with a T ½ life of 4.8±0.8 hrs. Incontrast Co-hArgI () displayed a bi-phasic loss of activity with anapparent first T½ of 6.1±0.6 hrs followed by much longer second T ½ of37±3 hrs.

FIG. 6 is a graph showing HPLC traces of the 20 standard amino acidsincubated with either Co-hArgI (Top panel) or dialysis buffer (LowerPanel). Co-hArgI incubated with the 20 standard amino acids resulted inthe loss of a single peak at RT=12.3 min, matching that of L-Argininecontrols, and the appearance of a single new peak at RT=18.8 min,matching that of L-Ornithine controls.

FIGS. 7A-B are graphs showing survival of HCC in tissue culture whentreated with various arginase variants (along with controls). FIG. 7Ademonstrates the survival of HCC tissue culture (Hep3b) when treatedwith 0-100 nM Arginase (Day 5). Mn-hArgI (▴), resulted in an apparentIC₅₀ of 5±0.3 nM (˜0.18 μg/ml). Incubations with Co-hArgI () lead to a15-fold increase in cytotoxicity with an apparent IC₅₀ of 0.33±0.02 nM(˜0.012 μg/ml). FIG. 7B is a graph showing the effect hArgI on thegrowth A375 melanoma cells (Day 5). Mn-hArgI (▴), resulted in anapparent IC₅₀ of 4.1±0.1 nM (˜0.15 μgimp. Incubation with Co-hArgI ()lead to a 13-fold increase in cytotoxicity with an apparent IC50 of0.32±0.06 nM (˜0.012 μg/ml).

FIG. 8 is a photograph of a non-denaturing electrophoretic gel showingthat the hArgI-E256Q variant is monomeric as opposed to trimericwild-type h-Argl.

FIG. 9 is a representative graph of steady-state kinetics of L-argininehydrolysis by Co-hArg-II () and Mn-hArg-II (∘). Cobalt substitutedhArg-II () hydrolysis of L-Arg at pH 7.4 and 37° C., with a k_(cat) of182±7 s⁻¹, a K_(M) of 126±18 μM, and a k_(cat)/K_(M) of 1,440±260 mM⁻¹s⁻¹. Manganese substituted hArg-II (∘) hydrolysis of L-Arg at pH 7.4 and37° C., with a k_(cat) of 48±2 s⁻¹, a K_(m) of 2,900±300 μM, andk_(cat)/K_(M) of 17±2 mM⁻¹ s⁻¹.

FIG. 10 is a graph showing serum L-arginine depletion in the mousemodel. Serum L-Arg concentrations of Balb/c mice treated with a singleIP dose of Co-hArgI are kept ≦ to 3-4 μM for over 3 days.

FIG. 11 is a graph showing HCC tumor xenograft reduction when treatedwith Co-hArgI as compared to controls. Nude mice bearing a Hep3b tumorxenografts were treated twice by IP injection with either PBS (∘) orCo-hArgI () at day 9 and at day 12. Tumor shrinkage was observed in themice treated with Co-hArgI whereas PBS treated tumors grew unchecked.

FIG. 12 14-20% SDS-PAGE showing hArgI conjugated to PEG MW 5000, with anapparent MW of ˜150 kDa.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The invention generally relates to compositions and methods for thetreatment of cancer with enzymes that deplete L-Arginine in serum. Insome embodiments, the cancer is one that does not express, or isotherwise deficient in, argininosuccinate synthetase (ASS), ornithinetranscarbamylase (OTC), or other enzymes required for argininebiosynthesis. Both native and mutated enzymes are contemplated, as wellas enzymes with modified metal cofactors, enzymes fused to otherpolypeptides as well as enzymes conjugated to polymers that increaseserum persistence, e.g., high molecular weight polyethylene glycol

I. ARGINASE

Arginase is a manganese-containing enzyme. It is the final enzyme of theurea cycle. Arginase is the fifth and final step in the urea cycle, aseries of biophysical reactions in mammals during which the bodydisposes of harmful ammonia. Specifically, arginase converts L-arginineinto L-ornithine and urea.

L-Arginine is the nitrogen donating substrate for nitric oxide synthase(NOS), producing L-Citrulline and NO. Although the K_(M) of Arginase(2-5 mM) has been reported to be much higher than that of NOS forL-Arginine (2-20 μM), Arginase may also play a role in regulating NOSactivity. Under certain conditions Arginase I is Cys-S-nitrosylated,resulting in higher affinity for L-Arginine and reduced availability ofsubstrate for NOS.

Arginase is a homo-trimeric enzyme with an α/β fold of a paralleleight-stranded β-sheet surrounded by several helices. The enzymecontains a di-nuclear metal cluster that is integral to generating ahydroxide for nucleophilic attack on the guanidinium carbon ofL-Arginine. The native metal for Arginase is Mn²⁺. These Mn²⁺ ionscoordinate water, orientating and stabilizing the molecule and allowingwater to act as a nucleophile and attack L-arginine, hydrolyzing it intoornithine and urea.

Mammals have two Arginase isozymes (EC 3.5.3.1) that catalyze thehydrolysis of L-Arginine to urea and L-Ornithine. The Arginase I gene islocated on chromosome 6 (6q.23), is highly expressed in the cytosol ofhepatocytes, and functions in nitrogen removal as the final step of theurea cycle. The Arginase II gene is found on chromosome 14 (14q.24.1).Arginase II is mitochondrially located in tissues such as kidney, brain,and skeletal muscle where it is thought to provide a supply ofL-Ornithine for proline and polyamine biosynthesis (Lopez et al., 2005).

Arginases have been investigated for nearly 50 years as a method fordegrading extracellular L-Arginine (Dillon et al., 2002). Some promisingclinical results have been achieved by introducing Arginase bytranshepatic arterial embolisation; following which, several patientsexperienced partial remission of HCC (Cheng et al., 2005). However,since Arginase has a high K_(M) (˜2-5 mM) and exhibits very low activityat physiological pH values, high dosing is required for chemotherapeuticpurposes (Dillon et al., 2002). While native Arginase is cleared fromcirculation within minutes (Savoca et al., 1984), a single injection ofPEG-Arginase MW5000 in rats was sufficient to achieve near completearginine depletion for ˜3 days (Cheng et al., 2007).

Cheng et al. made the surprising observation that many human HCC cellslines do not express OTC (in addition to ASS) and thus they aresusceptible to PEG-Arginase (Cheng et al., 2007). In mice implanted withHep3b hepatocarcinoma cells weekly administration of PEG-Arginaseresulted in tumor growth retardation which was accentuated byco-administration of 5-fluorouracil (5-FU). However, PEG-Arginase wasused at the very high doses that are impractical for human therapy,reflecting its lower physiological activity.

To address these issues a bacterial arginine hydrolyzing enzyme,Arginine Deiminase or ADI which displays good kinetics and stability hasbeen tested in vitro. A PEGylated form of ADI is now undergoing PhaseII/III clinical trials. Unfortunately ADI is a bacterial enzyme andtherefore it induces strong immune responses and adverse effects in mostpatients. However, for those patients that do not develop significantadverse responses, an impressive percentage exhibit stable disease orremission. Nonetheless because of its unfavorable immunological profileit is unlikely that L-Arginine depletion by ADI will become a mainstreamtreatment for liver cancer.

For clinical use, it is essential that the arginase is engineered toallow it to persist for long times (e.g., days) in circulation. In theabsence of any modification, human arginase has a half life of only afew minutes in circulation primarily because its size is notsufficiently large to avoid filtration though the kidneys. Unmodifiedhuman Arginase is very susceptible to deactivation in serum and it isdegraded with a half life of only four hours. Therefore, the presentinvention developed novel and improved forms of arginase for clinicalresearch and potential therapeutic use with improved circulationpersistence.

II. ARGINASE VARIANTS

Mammals have two Arginase isozymes (EC 3.5.3.1) that catalyze thehydrolysis of L-Arginine to urea and L-Ornithine. The Arginase I gene islocated on chromosome 6 (6q.23), is highly expressed in the cytosol ofhepatocytes, and functions in nitrogen removal as the final step of theurea cycle. The Arginase II gene is found on chromosome 14 (14q.24.1).Arginase II is mitochondrially located in tissues such as kidney, brain,and skeletal muscle where it is thought to provide a supply ofL-Omithine for proline and polyamine biosynthesis (Lopez et al., 2005).

L-Arginine is the sole substrate for nitric oxide synthase (NOS),producing L-Citrulline and NO. Although the KM of Arginase (2-5 mM) hasbeen reported to be much higher than that of NOS for L-Arginine (2-20μM), Arginase may also play a role in regulating NOS activity (Duranteet al., 2007). Under certain conditions Arginase I isCys-S-nitrosylated, resulting in higher affinity for L-Arginine andreduced availability of substrate for NOS (Santhanam et al., 2007).Arginase is a homo-trimeric enzyme with an α/β fold of a paralleleight-stranded β-sheet surrounded by several helices. The enzymecontains a di-nuclear metal cluster that is integral to generating ahydroxide for nucleophilic attack on the guanidinium carbon ofL-Arginine (Cama et al., 2003; Dowling et al., 2008). The native metalfor Arginase is Mn²⁺. Arginase with the native metal (ie. Mn2+) exhibitsa pH optimum of 9. At physiological pH the enzyme exhibits more than a10-fold lower k_(cat)/K_(m). in the hydrolysis of L-Arginine (FIG. 4).The low catalytic activity displayed by the authentic human arginasewith the native Mn²⁺ enzyme presents a problem for human therapy sinceit means that impractical doses of the enzyme have to be used to achievea therapeutically relevant reduction in L-Arginine plasma levels.

In some aspects, the present invention contemplates mutant arginaseswherein the natural metal cofactor (Mn²⁺) is replaced with anothermetal. It has been found that substitution of the metal cofactor inhuman arginase exerts a beneficial effect on the rate of hydrolysis ofL-Arginine and stability under physiological conditions when compared tonative human arginase with the natural metal cofactor. The substitutionof the native metal (Mn²⁺) with other divalent cations can be exploitedto shift the pH optimum of the enzyme to a lower values and thus achievehigh rates of L-arginine hydrolysis under physiological conditions.Human Arginase I and II proteins of the present invention have twoMn(II) sites; therefore, either or both sites can be substituted so asto generate a mutatated Arginase I or II protein with a non-native metalcofactor.

In some embodiments, the metal is cobalt (Co²⁺). Incorporation of Co2+in the place of Mn²⁺ in human Arginase I or human Arginase II results indramatically higher activity at physiological pH. It was found that anenzyme containing Co′ (“Co-hArgI”) displayed a 10 fold increase ink_(cat)/K_(M) in vitro at pH 7.4, which in turn translated into a 15fold increase in HCC cytotoxicity and a 13-fold increase in melanomacytotoxity as compared to the human Arginase I which contains Mn²⁺. Itwas also found that a pharmacological preparation of Co-hArgI couldclear serum L-Arg for over 3 days in mice with a single injection.Furthermore, it was found that a pharmacological preparation of Co-hArgIcould shrink HCC tumor xenografts in nude mice whereas Mn-hArgI onlyslowed tumor growth (Ensor et aL, 2002).

In some embodiments, the present invention provides a human arginaseprotein comprising at least one amino acid substitution at the metalbinding site. The structure of Arginase shows an active site cleftcontaining two Mn²⁺ ions, with the more deeply localized ion designatedMnA coordinated to H101, D124, D128, D232 and bridging hydroxide. Theother metal is designated Mn_(B) and is coordinated by H126, D124, D232,D234 and bridging hydroxide (Christianson and Cox, 1999). The residuescomprising the metal binding site for the first shell of Arginase I areH101, D124, H126, D128, D232, and D234 and for the second shell areW122, D181, and 5230. Similarly, the residues comprising the metalbinding site for the first shell of Arginase II are H120, D143, 11145,D147, D251, D253 and for the second shell are W141, D200, S249.

Arginase has been shown to require both Mn²⁺ ions for full activity,however MnA can be reversibly dissociated resulting in an enzyme withhalf its catalytic activity (Scolnick et al., 1997). Metal (A) of hArgIis coordinated to the imidazole of H101, which is in turn hydrogenbonded to the hydroxyl of S230. Metal (B) of hArgI is coordinated to theimidazole of H126, which has a 2^(nd) shell hydrogen bond with thecarboxyl of D181. The positions involved in the binding of the metalwere subjected to saturation mutagenesis and the resulting librarieswere screened using a microtiter well plate assay for arginase activity(described in more detail below in the examples) to isolate clonesexpressing proteins that display higher catalytic activity. Novel cloneswere identified by sequencing, re-transformed into E. coli (BL21) andpurified and kinetically characterized as described below in theexamples. Variants displaying apparent activity ≧ to wild-type werepurified in larger scale and assayed for their steady-state kineticparameters of k_(cat) & K_(M). The following variants were found to havegreater k_(cat)/K_(m) constants than Co-hArgI: D181S, D181E/S230A(double mutant containing two substitutions). Similarly the amino acidsubstitutions S230C and S230G were found to have a particularlyimportant effect on catalytic activity and also on serum stability.Additionally, amino acids removed from the metal binding site were alsosubjected to combinatorial saturation mutagenesis. For example, it wasfound that a C303P substitution in Co-hArg I conferred a 10-fold higherk_(cat)/K_(m) relative to the native Mn-hArg I at pH 7.4. Many of thesevariant or mutant forms of the arginase are contemplated for use in thetreatment of cancer, including where they are made as fusion proteins,e.g. with an Fc region (or portion thereof) of an immunoglobulin (inorder to increase half-life).

The Cys₃₀₃ variants were also tested for serum stability. It was foundthat a C303P variant, i.e. a single amino acid substitution in Arginase,exhibits a ˜60% increase in serum stability which in turn translatesinto a 30 fold increase in HCC cytotoxicity as compared to the Mnsubstituted enzyme at pH 7.4. In one embodiment, the present inventioncontemplates treatment with this novel enzyme or this novel enzyme withfurther mutations. In a particular embodiment, this novel enzyme isemployed for treatment as an Arginase-Fc protein fusion that capitalizeson the endosomal recycling of the IgG fc domain to ensure long serumpersistence of the Arginase variant. Long serum persistence improves theuse of Arginase as a therapeutic.

III. PEGYLATION

In certain aspects of the invention, methods and compositions related topegylated arginase are disclosed. Specifically, pegylation of arginaseat an engineered Cysteine residue (e.g., substituting the third residueof the N-terminal) may be used to produce a homogenous pegylatedarginase composition. Methods for isolation of pegylated arginase basedon temporary disruption of polymerization are also disclosed.

Pegylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. Pegylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity),increase the hydrodynamic size (size in solution) of the agent whichprolongs its circulatory time by reducing renal clearance. Pegylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step in pegylation is the suitable functionalization of thePEG polymer at one or both terminals. PEGs that are activated at eachterminus with the same reactive moiety are known as “homobifunctional”,whereas if the functional groups present are different, then the PEGderivative is referred as “heterobifunctional” or “heterofunctional.”The chemically active or activated derivatives of the PEG polymer areprepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, tyrosine. The N-terminal amino group and theC-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformatesand carbonates. In the second generation pegylation chemistry moreefficient functional groups such as aldehyde, esters, amides etc. madeavailable for conjugation.

As applications of pegylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acidsand NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule (as shown in the example with PEGbis-vinylsulfone).

Proteins are generally PEGylated at nucleophilic sites such asunprotonated thiols (cysteinyl residues) or amino groups. Examples ofcysteinyl-specific modification reagents include PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone. All four are stronglycysteinyl-specific under mild conditions and neutral to slightlyalkaline pH but each has some drawbacks. The amide formed with themaleimides can be somewhat unstable under alkaline conditions so theremay be some limitation to formulation options with this linker. Theamide linkage formed with iodo PEGs is more stable, but free iodine canmodify tyrosine residues under some conditions. PEG thiols formdisulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific pegylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl pegylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the pegylation reagent and is still biologicallyactive after pegylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the pegylation reaction difficult to control atlarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However; this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificpegylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the pegylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However; this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from pegylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of pegylation chemistry.

There are several parameters to consider when developing a pegylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofpegylation conditions can be very useful. For thiol-specific pegylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. (Oxygen can contributeto intermolecular disulfide formation by the protein, which will reducethe yield of the PEGylated product.) The same factors should beconsidered (with the exception of oxygen) for amine-specificmodification except that pH may be even more critical, particularly whentargeting the N-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the pegylationreaction. For example, if the pegylation agent is only 70 percentactive, the amount of PEG used should ensure that only active PEGmolecules are counted in the protein-to-PEG reaction stoichiometry. Howto determine PEG reactivity and quality will be described later.

IV. PROTEINS AND PEPTIDES

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such asstabilized arginase multimers. These peptides may be comprised in afusion protein or conjugated to an agent as described supra.

A. Proteins and Peptides

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide” and“peptide are used interchangeably herein.

In certain embodiments the size of at least one protein or peptide maycomprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about110, about 120, about 130, about 140, about 150, about 160, about 170,about 180, about 190, about 200, about 210, about 220, about 230, about240, about 250, about 275, about 300, about 325, about 350, about 375,about 400, about 425, about 450, about 475, about 500, about 525, about550, about 575, about 600, about 625, about 650, about 675, about 700,about 725, about 750, about 775, about 800, about 825, about 850, about875, about 900, about 925, about 950, about 975, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1750, about 2000,about 2250, about 2500 or greater amino acid residues.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine,β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid,piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelicacid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelicacid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsnN-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIleallo-Isoleucine MeGly N-Methylglycine, sarcosine MelleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

B. Nucleic Acids and Vectors

In certain aspects of the invention, nucleic acid sequences encoding afusion protein as a stabilized multimeric arginase may be disclosed.Depending on which expression system to be used, nucleic acid sequencescan be selected based on conventional methods. For example, humanarginase I and II contain multiple codons that are rarely utilized in E.coli that may interfere with expression, therefore the respective genesor variants thereof may be codon optimized for E. coli expression.Various vectors may be also used to express the protein of interest,such as a fusion multimeric arginase or a cysteine-substituted arginase.Exemplary vectors include, but are not limited, plasmid vectors, viralvectors, transposon or liposome-based vectors.

C. Host Cells

Host cells, preferably eukaryotic cells, useful in the present inventionare any that may be transformed to allow the expression and secretion ofarginase and fusion multimers thereof. The host cells may be bacteria,mammalian cells, yeast, or filamentous fungi. Various bacteria includeEscherichia and Bacillus. Yeasts belonging to the genera Saccharomyces,Kiuyveromyces, Hansenula, or Pichia would find use as an appropriatehost cell. Various species of filamentous fungi may be used asexpression hosts including the following genera: Aspergillus,Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,Endothia, Mucor, Cochliobolus and Pyricularia.

Examples of usable host organisms include bacteria, e.g., Escherichiacoli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al.,1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcuslividans (Hopwood et al., 1985); yeasts, e.g., Saccharomyces cerevisiaeAH 22 (Mellor et al., 1983) and Schizosaccharomyces pombe; filamentousfungi, e.g., Aspergillus nidulans, Aspergillus awamori (Ward, 1989),Trichoderma reesei (Penttila et al., 1987; Harkki et al, 1989).

Examples of mammalian host cells include Chinese hamster ovary cells(CHO-KI; ATCC CCL61), rat pituitary cells (GH₁; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548)SV40-transformed monkey kidney cells (COS-I; ATCC CRL 1650) and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing beingillustrative but not limitative of the many possible host organismsknown in the art. In principle, all hosts capable of secretion can beused whether prokaryotic or eukaryotic.

Mammalian host cells expressing the arginase and/or their fusionmultimers are cultured under conditions typically employed to culturethe parental cell line. Generally, cells are cultured in a standardmedium containing physiological salts and nutrients, such as standardRPMI, MEM, IMEM or DMEM, typically supplemented with 5-10% serum, suchas fetal bovine serum. Culture conditions are also standard, e.g.,cultures are incubated at 37° C. in stationary or roller cultures untildesired levels of the proteins are achieved.

D. Protein Purification

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure peptide are ion-exchangechromatography, gel exclusion chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatographyand isoelectric focusing. A particularly efficient method of purifyingpeptides is fast performance liquid chromatography (FPLC) or even highperformance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

In certain embodiments a protein or peptide may be isolated or purified,for example, a stabilized arginase multimeric fusion protein, or anarginase prior or post pegylation. For example, a His tag or an affinityepitope may be comprised in such a arginase variant to facilitatepurification. Affinity chromatography is a chromatographic procedurethat relies on the specific affinity between a substance to be isolatedand a molecule to which it can specifically bind. This is areceptor-ligand type of interaction. The column material is synthesizedby covalently coupling one of the binding partners to an insolublematrix. The column material is then able to specifically adsorb thesubstance from the solution. Elution occurs by changing the conditionsto those in which binding will not occur (e.g., altered pH, ionicstrength, temperature, etc.). The matrix should be a substance thatitself does not adsorb molecules to any significant extent and that hasa broad range of chemical, physical and thermal stability. The ligandshould be coupled in such a way as to not affect its binding properties.The ligand should also provide relatively tight binding. And it shouldbe possible to elute the substance without destroying the sample or theligand.

Size exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeation chromatographywhich is used when an organic solvent is used as a mobile phase.

The underlying principle of SEC is that particles of different sizeswill elute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size should elute together. Eachsize exclusion column has a range of molecular weights that can beseparated. The exclusion limit defines the molecular weight at the upperend of this range and is where molecules are too large to be trapped inthe stationary phase. The permeation limit defines the molecular weightat the lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

High-performance liquid chromatography (or High pressure liquidchromatography, HPLC) is a form of column chromatography used frequentlyin biochemistry and analytical chemistry to separate, identify, andquantify compounds. HPLC utilizes a column that holds chromatographicpacking material (stationary phase), a pump that moves the mobilephase(s) through the column, and a detector that shows the retentiontimes of the molecules. Retention time varies depending on theinteractions between the stationary phase, the molecules being analyzed,and the solvent(s) used.

V. PHARMACEUTICAL COMPOSITIONS

It is contemplated that the novel arginases of the present invention canbe administered systemically or locally to inhibit tumor cell growthand, most preferably, to kill cancer cells in cancer patients withlocally advanced or metastatic cancers. They can be administeredintravenously, intrathecally, and/or intraperitoneally. They can beadministered alone or in combination with anti-proliferative drugs. Inone embodiment, they are administered to reduce the cancer load in thepatient prior to surgery or other procedures. Alternatively, they can beadministered after surgery to ensure that any remaining cancer (e.g.cancer that the surgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, as injectables. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired the compositions maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, stabilizing or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, pharmaceutical compositions of the presentinvention comprise an effective amount of one or more arginase variantsor additional agent dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of an pharmaceutical composition thatcontains at least one arginase variant, such as a stabilized multimericarginase or a pegylated arginase isolated by the method disclosedherein, or additional active ingredient will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18^(th) Ed., 1990, incorporatedherein by reference. Moreover, for animal (e.g., human) administration,it will be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18^(th) Ed., 1990, incorporated herein by reference). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the pharmaceutical compositions is contemplated.

The present invention may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18^(th) Ed., 1990, incorporatedherein by reference).

The arginase variants may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include arginasevariants, one or more lipids, and an aqueous solvent. As used herein,the term “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds are well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compoundswhich contain long-chain aliphatic hydrocarbons and their derivatives. Alipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof. Of course, compounds other than those specificallydescribed herein that are understood by one of skill in the art aslipids are also encompassed by the compositions and methods of thepresent invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the stabilized multimeric arginase or pegylatedarginase may be dispersed in a solution containing a lipid, dissolvedwith a lipid, emulsified with a lipid, mixed with a lipid, combined witha lipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

VI. DEFINITIONS

The term “aa” refers to amino acid(s). Amino acid substitutions areindicated by the amino acid position, e.g. 303, in the molecule using aletter code (the letter in front of the number indicates the amino acidbeing replaced, while the letter after the number indicates the aminoacid being introduced).

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (i.e., a human arginase or variantthereof) joined (or operably linked) to an exogenous protein fragment(the fusion partner which consists of a non-arginase protein). Thefusion partner may enhance serum half-life, solubility, or both. It mayalso provide an affinity tag (e.g. his-tag) to allow purification of therecombinant fusion protein from the host cell or culture supernatant, orboth.

The terms “in operable combination”, “in operable order” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired protein molecule isproduced. The term also refers to the linkage of amino acid sequences insuch a manner so that a functional protein is produced.

The term “K_(m)” as used herein refers to the Michaelis-Menton constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term k_(cat) as used herein refers to the turnover number or thenumber of substrate molecule each enzyme site converts to product perunit time, and in which the enzyme is working at maximum efficiency.

The term Kcat/Km as used herein is the specificity constant which is ameasure of how efficiently an enzyme converts a substrate into product.

The term “Mn-hArgI” refers to human Arginase I with an Mn(II) cofactor.The term “Co-hArgI” refers to human Arginase I (mutant or native) with aCo(II) cofactor.

The term “IC₅₀” is the half maximal (50%) inhibitory concentration (IC)and thus a measure of effectiveness.

The term “pegylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. (Harris et al.,2001). Attachment to various drugs, proteins, and liposomes has beenshown to improve residence time and decrease toxicity. (Greenwald etal., 2000; Zalipsky et al., 1997). PEG can be coupled (e.g. covalentlylinked) to active agents through the hydroxyl groups at the ends of thechain and via other chemical methods; however, PEG itself is limited toat most two active agents per molecule. In a different approach,copolymers of PEG and amino acids have been explored as novelbiomaterials which would retain the biocompatibility properties of PEG,but which would have the added advantage of numerous attachment pointsper molecule (providing greater drug loading), and which can besynthetically designed to suit a variety of applications (Nathan et al.,1992; Nathan et al., 1993).

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired enzymatic activity is retained. The term “subject” refers toanimals, including humans.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “variant” or “mutant” refers to a gene or gene productwhich displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

VII. KITS

The present invention provides kits, such as therapeutic kits. Forexample, a kit may comprise one or more pharmaceutical composition asdescribed herein and optionally instructions for their use. Kits mayalso comprise one or more devices for accomplishing administration ofsuch compositions. For example, a subject kit may comprise apharmaceutical composition and catheter for accomplishing directintravenous injection of the composition into a cancerous tumor. Inother embodiments, a subject kit may comprise pre-filled ampoules of astabilized multimeric arginase or isolated pegylated arginase,optionally formulated as a pharmaceutical, or lyophilized, for use witha delivery device.

Kits may comprise a container with a label. Suitable containers include,for example, bottles, vials, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer may hold a composition which includes an antibody that iseffective for therapeutic or non-therapeutic applications, such asdescribed above. The label on the container may indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above. The kit of the invention willtypically comprise the container described above and one or more othercontainers comprising materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

VIII. EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof. In the experimental disclosure whichfollows, the following abbreviations apply: eq (equivalents); M (Molar);μM (micromolar); mM (millimolar); N (Normal); mol (moles); mmol(millimoles); pmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); EC (degrees Centigrade); MW (molecular weight); PBS(phophate buffered saline); min (minutes).

Example 1 Gene Synthesis and Expression of Human Arginase I & II

The human Arginase I and II genes both contain mutiple codons that arerarely utilized in E. coli that can interfere with expression. Thus, inorder to optimize protein expression in E. coli, the respective geneswere assembled with codon optimized oligonucleotides designed withDNA-Works software (Hoover et aL, 2002). Each construct contains anN-terminal Ncol restriction site, an in-frame N-terminal His₆ tagfollowed by a Tobacco Etch Virus (TEV) protease site and a C-terminalBamHI site for simplifying cloning. Cleavage by TEV protease removes theHis6 peptide and the N-terminal Met of arginase. An Arginase II gene wasdesigned with a TEV protease cleavage site and without the first native21 aa. The first 21 aa are a putative mitochondrial-targeting sequenceand its removal results in greater protein yield and stability(Colleluori et aL, 2001). After cloning into a pET28a vector (Novagen),E. coli (BL21) containing an appropriate Arginase expression vector weregrown at 37° C. using Terrific Broth (TB) media containing 50 μg/mlkanamycin in shake flasks at 250 rpm until reaching an OD₆₀₀ of 0.5-0.6.At that point the cultures were transferred to 25° C., induced with 0.5mM IPTG and allowed to express protein for an additional 12 hrs. Cellpellets were then collected by centrifugation and re-suspended in anIMAC buffer (10 mM NaPO₄/10 mM imidazole/300 mM NaCl, pH 8). After lysisby a French pressure cell, lysates were centrifuged at 20,000×g for 20mM at 4° C., and the resulting supernatant was applied to a cobalt ornickel IMAC column, washed with 10-20 column volumes of IMAC buffer, andthen eluted with an IMAC elution buffer (50 mM NaPO₄/250 mMimidazole/300 mM NaCl, pH 8). The desired divalent metal cation isincorporated by incubation with 10 mM metal (CoCl₂ or MnSO₄) for 15 mMat 50°-55° C., followed by filtration through a 0.45 gm syringe filter.Using a 10,000 MWCO centrifugal filter device (Amicon), proteins werethen buffer-exchanged several times into a 100 mM Hepes, 10% glycerol,pH 7.4 solution. Aliquots of Arginase enzyme were then flash frozen inliquid nitrogen and stored at −80° C. Arginase purified in this manneris >95% homogeneous as assessed by SDS-PAGE and coomassie staining (FIG.2). The yield is calculated to be 200 mg/L culture based upon thecalculated extinction coefficient, ε₂₈₀=24,180 M⁻¹ cm⁻¹ in a finalbuffer concentration of 6 M guanidinium hydrochloride, 20 mM phosphatebuffer, pH 6.5 (Gill and von Hippel, 1989).

Example 2 Incorporating and Determining Metal Content in Arginase I

As mentioned in Example 1, incorporation of Mn²⁺ and Co²⁺ can beachieved by purifying Arginase, followed by an incubation step with 10mM metal at 50° C. for 10 min. In order to determine the final metalcontent and identity of the Arginase preps, protein samples of Mn-hArgI(145 μM), Co-hArgI (182 μM) and associated dialysis buffers (100 mMHepes, pH 7.4) were diluted in 2% nitric acid and analyzed byinductively coupled plasma mass spectrometry (ICP-MS, Department ofGeological Sciences, University of Texas at Austin) to quantify theprotein's cobalt, iron, manganese and zinc content by subtracting theconcentration of metals found in dialysis buffer from the metalconcentration of the final protein samples and dividing by proteinconcentration. To determine protein concentrations, an extinctioncoefficient was calculated for hArgI based on amino acid sequence (Gilland von Hippel, 1989). All protein concentrations for Arginase I werecalculated based upon the calculated ε₂₈₀=24,180 M⁻¹ cm⁻¹ in a finalbuffer concentration of 6 M guanidinium hydrochloride, 20 mM phosphatebuffer, pH 6.5. For comparison, Arginase concentration was alsocalculated by BCA assay using dilutions of BSA as a standard. Using thismethod it was found that Arginase samples incubated with Co²⁺ contain2.1±0.5 equivalents Co and 0.4±0.1 equivalents Fe, with no detectableamounts of Zn or Mn. Samples incubated with Mn²⁺ contain 1.5±0.2equivalents Mn and 0.4±0.1 equivalents Fe, and no detectable amounts ofZn or Co. Thus, heat incubation is an efficient method for incorporationof Cobalt.

Example 3 Incorporating and Determining Metal Content in Arginase II

Efficient metal incorporation into Arginase II was achieved by culturingE. coli harboring the ArgII gene in minimal media until an OD₆₀₀ of 1 isreached, whereupon the protein was expressed with 1 mM IPTG and 100 μMCoCl₂ for an additional 12 hrs. In order to determine the final metalcontent and identity of the Arginase preps, protein samples of Co-hArgI(290 μM) and associated dialysis buffers (100 mM Hepes, pH 7.4) werediluted in 1% nitric acid and analyzed by inductively coupled plasmamass spectrometry (ICP-MS, Department of Geological Sciences, Universityof Texas at Austin) to quantify the protein's cobalt, iron, manganeseand zinc content by subtracting the concentration of metals found indialysis buffer from the metal concentration of the final proteinsamples and dividing by protein concentration. To determine proteinconcentrations, an extinction coefficient was calculated for hArgI basedon amino acid sequence (Gill and von Hippel, 1989). All proteinconcentrations for Arginase II were calculated based upon the calculatedε₂₈₀=22,900 M⁻¹ cm⁻¹ in a final buffer concentration of 6 M guanidiniumhydrochloride, 20 mM phosphate buffer, pH 6.5. For comparison, Arginaseconcentration was also calculated by BCA assay using dilutions of BSA asa standard. Using this method it was found that Arginase samplesexpressed with Co²⁺ contain 1.35±0.1 equivalents Co and 0.63±0.1equivalents Fe, with no detectable amounts of Zn or Mn.

Example 4 Steady State Kinetics of Cobalt Arginase I at Physiological pH

Diacetylmonoxine (DAMO) dervitization of urea products in the presenceof strong acids, thiosemicarbazide and Fe³⁺ with heating to produce achromophore with a λ_(max) of ˜530 nm, was used to monitor ureaproduction following hydrolysis of L-Arginine by arginase. The dyestructure is not definitively known, but the reaction is hypothesized tobe a condensation of DAMO and urea/uriedo that is possibly stabilized byFe³⁺ ions (Beale and Croft, 1961). A standard curve of urea vs. A₅₃₀ wasconstructed that was found to be linear between 0-300 μM urea with alower detection limit of 1-2 μM. The steady-state kinetics of Co-hArgI,and Mn-hArgI were examined over a range of L-arginine concentrations(0-40 mM) in a 100 mM Hepes buffer pH 7.4, 37° C. Typically reactionswere performed by equilibrating 200 μL in 1.5 ml eppendorf tubes at 37°C. in a heat block, starting the reaction by adding 5 μL of enzyme for30 sec and quenching with 15 μL of 12 N HCl. Reactions and blanks werethen mixed with 800 μL of color developing reagent (COLDER) (Knipp andVasak, 2000) and boiled for 15 min. After cooling for 10 min, thesamples were transferred to cuvettes and read at 530 nm on aspectrophotometer. L-Arginine has a background absorbance that makescorrection necessary, so L-Arginine blanks were included for allconcentrations used. The resulting data is then corrected for backgroundand the concentrations of product formed calculated from the standardcurve. The product is then divided by the time and the concentration ofenzyme used and v_(o)/[E] is plotted vs. substrate concentration and fitdirectly to the Michaelis-Menten equation (FIG. 3), wherev_(o)/[E]=k_(cat)*[S]/([S]+K_(M)). With this method, Co-hArgI had ak_(cat) of 240±14 s⁻¹, a K_(M) of 190±40 μM, and k_(cat)/K_(M) of1,270±330 mM⁻¹ s⁻¹,” as compared to Mn-hArgI, which had a k_(cat) of300±12 s⁻¹, a K_(M) of 2,330±260 μM, and k_(cat)/K_(M) of 129±20 mM⁻¹s⁻¹. The use of Cobalt as a cofactor at physiological pH leads to 10fold increase in the specificity constant.

Example 5 Steady State Kinetics of Cobalt Arginase II at PhysiologicalpH

Arginase II purified as described in Example 3 and characterized asdescribed in Example 4 was found to have a k_(cat) of 182±7 s⁻¹, a K_(M)of 126±18 μM, and a k_(cat)/KM of 1,440±260 mM⁻¹ s⁻¹ as compared toMn-hArgII where we found a k_(cat) of 48±2 s⁻¹, a K_(m) of 2,900±300 μM,and k_(cat)/Km of 17±2 mM⁻¹ s⁻¹. The use of Cobalt as a cofactor atphysiological pH leads to 80 fold increase in the specificity constantfor Arginase II.

Example 6 96-Well Plate Screen for Arginase Activity and Ranking Clones

Arginase hydrolysis of L-Arginine produces L-Ornithine and urea. TheL-Arginine hydrolysis assay of Example 3 was adapted to 96-well plateformat for the detection of urea and used for screening libraries ofprotein mutants and for rank-ordering clones with the greatest Arginaseactivity. Clones displaying greater than 2-fold increase in activitywere selected for further characterization. The assay was shown to havea dynamic range of ˜5-200 μM for ureido product detection. More than 500clones can easily be screened per day via manual screening. The signaloutput, i.e., color intensity, reflects three main parameters, namelythe specificity constant (k_(cat)/K_(M)), the enzyme concentration[Enz], and time (t). If necessary, enzyme levels in individual clonescan be detected by ELISA; however, generally enzyme expression ofArginase varies less than two-fold and therefore expression differencesdo not constitute a significant issue.

Single colonies were picked into 96-well culture plates containing 75 μLof TB media/well containing 50 μg/ml kanamycin. These cultures were thengrown at 37° C. on a plate shaker until reaching an OD₆₀₀ of 0.8-1,cooled to 25° C., whereupon an additional 75 μL of media containing 50μg/ml kanamycin, and 0.5 mM IPTG was added. Expression was performed at25° C. with shaking for ˜2 hrs, following which 100 μL of culture/wellwas transferred to a 96 well assay plate. The assay plates were thencentrifuged to pellet the cells, the media was removed, and the cellswere lysed by addition of 50 μL/well of B-PER protein extraction reagent(Pierce). An additional 50 μL/well of 200 μM L-Arginine, 1 mM CoCl₂, ina 100 mM HEPES buffer, pH 7.4 was subsequently added and allowed toreact at 37° C. After reacting ˜1-2 mM, 100 μL/well of color developingreagent are added and the plate was processed (Knipp and Vasak, 2000).Colonies having the ability to produce urea resulted in formation of abright red dye with a λ_(max) of 530 nm.

Example 7 pH Rate Dependence of Cobalt Arginase and Manganese Arginase

To examine the pH dependence of k_(cat)/K_(M) of cobalt and manganesesubstituted Arginase, the steady-state kinetic constants were determinedacross a broad range of pH values. The following buffers were used:sodium acetate (pH 5-5.5), MES (pH 6-6.5), HEPES (pH 7-7.8), Tris (pH8-9), Capso (pH 9-10.5), all at a 100 mM concentration. All kineticswere determined in at least triplicate at 37° C. After fitting thekinetic data to the Michaelis-Menten equation, the k_(cat)/K_(M) valueswere calculated and fit to a Henderson-Hasselbach equation to determinepK_(a) values. Because fits to two pK_(a) values closer than 3.5 unitstend to underestimate y_(max), Segel's method was used to calculatecorrected pK_(a) values for each limb of the k_(cat)/K_(M) profiles(Segel, 1975). Adjusted fits of k_(cat)/K_(m) vs. pH resulted in a bellshaped curve with Co-hArgI having an ascending limb pK_(a) of 7.5±0.1and a descending limb pK_(a) of 9.8±0.1. Mn-hArgI also had a bell shapedcurve with an ascending limb pK_(a) of 8.5±0.1 and a descending limbdisplaying an apparent pK_(a) value of 10.1±0.1 (FIG. 4). Mn-hArgI andCo-Argl enzymes exhibited a A pK_(a) of 1 pH units. This shift in pK_(a)upon Co substitution likely imparts much of the observed improvement inthe specificity constant. At physiological pH, approximately 44% ofCo-hArgI would have hydroxide bound as opposed to 7% with Mn-hArgI.

Example 8 The Effect of Mutations at Position 303

An NNS codon saturation library at position 303 was constructed andscreened using the following mutagenic primers: Forward′5-cgatcacgttagcaNNSttcggtttagcccg (SEQ ID NO:3), and reverse′5-CGGGCTAAACCGAAsnnTGCTAACGTGATCG (SEQ ID NO:4), using the hArgI geneas template DNA and specific end primers; forward′5-GATATACCATGGGTTCTTCTCACCATCATCACCACCACAGCTCTGGCG (SEQ ID NO:5) and;reverse ′5-CGAATTCGGATCCTCACTTCGGTGGATTCAGATAATCAATT (SEQ ID NO:6). ThePCR product digested with Ncol and BamHI and ligated into pET28a vectorwith T4 DNA ligase. The resulting ligation was transformed directly intoE. coli (BL21), plated on LB-kanamycin plates for subsequent screeningas described in Example 4. Clones exhibiting highest activity wereisolated and the DNA was sequenced. The respective enzyme variants werepurified as described in Example 1 and heat incubated with Cobalt asdescribed in Example 2. All proteins were purified to >95% homogeneityas assessed by SDS-PAGE. Arginine hydrolysis kinetics were determinedwith a range of L-arginine concentrations (0-2 mM) at 37° C. in a 100 mMHepes buffer pH 7.4, and the resulting data fit to the Michaelis-Mentenequation in Kaleidagraph. Cys₃₀₃ substituted with Phe or Ile lead to a 2fold & 1.6 fold increase in k_(cat)/K_(m) respectively as compared towild-type Co-hArgI. Leu, Pro, His, and Arg substitutions had about 90%of wild-type activity.

The Cys₃₀₃ variants were also tested for serum stability at 37° C. asfollows: Purified enzymes were added to pooled human serum (Innovative)at a concentration of 1 μM and incubated at 37° C. Every ˜24 hours,aliquots were withdrawn and tested in triplicate for their ability tohydrolyze 1 mM L-arginine in a 100 mM Hepes buffer pH 7.4. After ˜4 daysthe resulting data was fit to either an exponential or logistic decaymodel to calculate T_(1/2) values. The stability of the wild-type enzymewas used as a standard and was calculated to be a T_(1/2) of 33±3 hrs.Enzymes substituted with Phe, Ile, Leu, and His were only about half asstable as wild-type. Mn-hArgI (0) displayed an exponential loss ofactivity with a T ½ life of 4.8±0.8 hrs. In contrast Co-hArgI (6)displayed a bi-phasic loss of activity with an apparent first T′/2 of6.1±0.6 hrs followed by much longer second T ½ of 37±3 hrs.

Example 9 Substrate Specificity

The selectivity of the engineered human Arginase for the hydrolysis ofarginine was evaluated. Co-hArgI (1 μM) or dialysis buffer was incubatedwith all 20 as (5 mM each) for 12 hr at 37° C. in a 220 mM phosphatebuffer pH 7.4. Standards, controls and experiments were derivatized withOPA and FMOC (Agilent) and separated on a C18 reverse phase HPLC column(Agilent) (5 μm, 4.6×150 mm) essentially as described by AgilentTechnologies (Publication Number: 5980-3088) except for modification ofthe separation protocol slightly by reducing the flow rate by ½ anddoubling the acquisition time to get better peak separation. The 20standard amino acids incubated with dialysis buffer showed 20 peaks withgood resolution, and the 20 standard amino acids incubated with Co-hArgIshowed 20 peaks (FIG. 6) with disappearance of the L-Arginine peak(RT=12.3 min) and appearance of one new peak with a retention timematching that of L-Ornithine (RT=18.8 min). None of the other aminoacids were observed to be affected by Co-hArg I.

Example 10 High Throughput Purification and Kinetic Screening ofVariants

A small-scale purification scheme was developed to rapidly purify dozensof proteins at once and carry out high throughput enzyme kineticsanalysis. 50 ml cultures of hArg I were expressed in 125 ml shake flasksas described in Example 1. 5 ml of the resulting culture were collectedby centrifugation. The cell pellets were then lysed with 400 μL of B-PERprotein extraction reagent (Pierce). The soluble fraction was mixed with500 μL IMAC lysis buffer and 100 μL of IMAC beads in a 1.5 ml Eppendorftube, incubated for two minutes and centrifuged at 3000 rpm for 20 s ina table top centrifuge. The supernatant was discarded and the beads arewashed with 2×1 ml IMAC lysis buffer by mixing/centrifugation anddiscarding the supernatant. hArg I was then eluted from the beads byaddition of 300 μL of IMAC elution buffer and another centrifuge step.The resulting hArg I containing supernatant was subjected to bufferexchange twice with a 100 mM Hepes, pH 7.4 buffer using a 10,000 MWCOcentrifugal concentration device (YM-10 Amicon). The protein was thenquantified by A₂₈₀, heated in the presence of Cobalt as above, and theresulting Co-hArg I was assessed by SDS-PAGE as described in Example 1.This method allows purification of 12-16 proteins in ˜2 hrs with a yieldof 200-300 μg protein at 90-95% purity as assessed by SDS-PAGE.

The enzyme variants were then tested for their ability to hydrolyzeL-Arginine by incubating 24 nM of enzyme with 200 μM of L-arginine inmicrotiter plate wells. Aliquots were collected at different time pointsand directly quenched into the acidic color-developing reagent (COLDER).After developing the dye and reading the absorption, the progress curvedata was fit to an exponential equation to estimate an apparentk_(cat)/K_(M) value.

Example 11 Engineering the 2^(nd) Shell Metal Ligands of Arginase forOptimal Activity

The catalytic power of a metallohydrolase stems in part from itsremarkable ability to depress the normal pK_(a) value of water (˜16) toa much lower value and coordinate the highly reactive hydroxide ion forattack on substrate. Both the kind of metal and its local environmentcomprising of 1^(st) and 2^(nd) shell ligands influence the pK_(a) ofthe nucleophilic water/hydroxide molecule (Christianson and Cox, 1999).Metal (A) of hArgI is coordinated to the imidazole of H101, which is inturn hydrogen bonded to the hydroxyl of S230. Metal (B) of hArgI iscoordinated to the imidazole of H126, which has a 2^(nd) shell hydrogenbond with the carboxyl of D181. An NNS codon saturation library atposition 181 and 230 was constructed using the following mutagenicprimers: (D181) Forward ′5-cattggcttacgtNNSgtcgacccagg (SEQ ID NO:7),reverse ′5-CCTGGGTCGACSNNACGTAAGCCAATG (SEQ ID NO:8); (S230) forward′5-cgtccaatccatctgNNSttcgatgttgacg (SEQ ID NO:9), reverse′5-CGTCAACATCGAASNNCAGATGGATTGGACG (SEQ ID NO:10), along with the hArgIgene and specific end primers via overlap extension PCR. After cloning,the library was transformed in E. coli (BL21) and screened as describedin Example 4. Novel clones were identified by sequencing, re-transformedinto E. coli (BL21) and purified and kinetically characterized asdescribed in Example 8. Variants displaying apparent activity ≧ towild-type were purified in larger scale and assayed for theirsteady-state kinetic parameters of k_(cat) & K_(M). The followingvariants were found to have greater k_(cat)/K_(m) constants thanCo-hArgI: hArg I D181S: 1420±200 s⁻¹ mM⁻¹, hArg I D181E/S230A: 1,450±200s⁻¹ mM⁻¹, hArg I S230C: 2,290±200 s⁻¹ mM⁻¹, and hArg I S230G: 2,340±70s⁻¹ mM⁻¹.

Example 12 Cytotoxicty of Co-Arg and its Variants Towards HepatocellularCarcinoma Cells and Metastatic Melanomas

In order to test the in vitro cytoxicity of engineered Arginase, varyingconcentrations (0-100 nM) of Mn-Argl, Co-Argl, or Co-hArgI variants wereincubated with HCC (Hep 3b) cells or melanoma (A375) cells (AmericanType Culture Collection) in 96-well plates at a seeding density of 500cells/well, in DMEM media supplemented with fetal bovine serum. After 24hours of incubation at 37° C., the cells were treated with Arginasecontaining media in triplicate at various concentrations. The controlsolution was a balanced salt solution in media. The treated cells weremaintained at 37° C. and 5% CO₂. Cells were tested by standard MTT assay(Sigma-Aldrich) on days 1, 3, 5, & 7 by addition of 100 μL/well of MTT(5 mg/mL), and incubated for 4 hours with gentle agitation one to twotimes per hour. Following this, the solution was aspirated and 200 μL ofDMSO was then added to each well. Absorbance at 570 nm was interpretedfor each well using an automated plate reader to determine the relativenumber of surviving cells compared to controls. The resulting data wasfit to an exponential equation to determine an apparent IC₅₀ value forArginase cytoxicity. FIG. 7A shows the effect of Mn-Argl, or Co-Argladdition on % MTT absorbance of HCC cells. The IC₅₀ values from day 5were calculated, yielding an IC₅₀ value for Mn-hArgI of 5±0.3 nM (˜0.18μg/ml) and a value of 0.33±0.02 nM for Co-hArgI (˜0.012 μg/ml). Thus,the Co-Argl enzyme appears to be 15 fold more cytotoxic than the Mnsubstituted enzyme against HCC. Against the metastatic melanoma cellline (A375) Mn-hArgI (FIG. 7B) resulted in an apparent IC₅₀ of 4.1±0.1nM (˜0.15 μg/ml). Incubation with Co-hArgI lead to a 13-fold increase incytotoxicity with an apparent IC₅₀ of 0.32±0.06 nM (˜0.012 μg/ml).

Example 13 Engineering an Fc-Arginase Fusion Protein for Enhanced InVivo Half-Life

Fusion to the IgG Fe domain has been employed extensively for prolongingthe in vivo half-lives of therapeutic polypeptides such as the TNF-αinhibitor etanercept (Enbril®). The Fc domain binds to the FcγRnreceptor, which is expressed on vascular endothelium and many othertissues (Roopenian and Akilesh, 2007). The affinity of FcγRn for the IgGFc domain is strongly pH dependent. Binding occurs at the acidic pH ofendosomal compartments allowing the protein to be recycled onto the cellsurface and thus escape proteolytic degradation. At the cell surface,the Fc domain is released from FcγRn because the binding affinity isvery low at physiological pH. Endosomal recycling via FcγRn is estimatedto increase the serum half-life of immunoglobulins at least 4-7 fold, toabout 7-14 days in humans. Fc fusions exploit this property to endowshort lived molecules with a long half life. However, the human Arginaseis a homotrimer and therefore if fused to the IgG Fc, which itself is adimer, the resulting Fc-Arginase polypeptide will likely form highmolecular weight aggregates.

This problem was avoided by employing mutant forms of Arginase thatdisrupt trimerization and are stable in the monomeric form. Thetrimerization and subunit interface of Arginase I have been studied insome detail (Lavulo et al., 2001). A single as substitution at Glu256Glnhas been shown to disrupt trimerization resulting in the formation ofmonomeric Arginase I enzyme (Sabio et al., 2001). This mutation wasintroduced into hArgI by the use of two mutagenic primers: Forward′5-ggtttaacgtatcgcCAGggcctgtatatcacgg (SEQ ID NO:11) and Reverse′5-CCGTGATATACAGGCCCTGGCGATACGTTAAACC (SEQ ID NO:12), and two specificend primers (Example 1) through overlap extension PCR, and cloning intoa pET28a vector. After expression and purification of this variant, thesteady-state kinetic analysis revealed nearly identical activitycompared to Co-hArgI with a k_(cat)/K_(M) of 1,320 s⁻¹ mM⁻¹. FIG. 8shows a non-denaturing PAGE gel showing that Co-hArgI-E256Q is amonomer, as expected.

This construct was then cloned into Fc expression vectors available. TheFc expression vector is a construct based on a pTRC99a plasmid(Amersham) that contains a DsbA leader sequence followed by the IgG Fccoding region, an EcoRI restriction site and a stop codon. The monomericArginase gene was placed in frame behind the Fc coding region bydigesting both vector and gene with EcoRI, and was subsequently ligatedand transformed into E. coli (BL21) for sequencing and expression. Sincethe IgG Fc is normally a glycosylated protein, expression of recombinantIgGs or of Fc fusions has so far been carried out in recombinantmammalian cells that, unlike bacteria, are capable of N-linkedglycosylation. However, while glycosylation at Asn297 is critical forthe binding to the activating and inhibitory Fcγ receptors (FcγRI-III inhumans) it does not have a noticeable effect on the affinity or pHdependent binding to FcγRn (Tao and Morrison, 1989; Simmons et al.,2002). Thus, aglycosylated IgG antibodies expressed in bacteria exhibitserum persistence in primates nearly indistinguishable from that offully glycosylated antibodies expressed in mammalian cells (Simmons etal., 2002). In contrast to prevailing earlier notions, IgG antibodiesand Fc proteins can be expressed efficiently in E. coli up to g/L levelsin fermenters. E. coli expression is technically much simpler andfaster. In addition, since the resulting protein is aglycosylated, itdoes not display glycan heterogeneity, an important issue in theexpression of therapeutic glycoproteins (Jefferis, 2007). The fusionprotein is purified by Protein A chromatography and the yield ofcorrectly folded, dimeric Fc-Arginase fusion relative to polypeptidesthat fail to dimerize is quantified by FPLC gel filtrationchromatography. This formulation has lead to a highly active and verystable form of human Arginase, suitable for in vivo trials.

Example 14 Pegylation of Arginase

Arginase was purified as described in Example 10 with one exception:after binding to the IMAC column, the protein was washed withextensively (80-90 column volumes) with an IMAC buffer containing 0.1%Triton 114 (This step removes most of the endotoxin), 10-20 columnvolumes of IMAC buffer, and then eluted with an IMAC elution buffer (50mM NaPO₄/250 mM imidazole/300 mM NaCl, pH 8). Arginase was bufferexchanged into a 100 mM NaPO₄ buffer at pH 8.3 using a 10,000 MWCOfiltration device (Amicon). Using a small reaction jar, Methoxy PEGSuccinimidyl Carboxymethyl Ester 5000 MW (JenKem Technology) was addedto Arginase at 40:1 molar ratio and allowed to react for 1 hr at 25° C.under constant stirring. The resulting mixture was then made 10 mM withCoCl₂ and heated at 50° C. for 10 minutes. After centrifuging to removeany precipitates, the PEG-5000 Arginase was extensively buffer exchanged(PBS with 10% glycerol) using a 100,000 MWCO filtration device (Amicon),and sterilized with a 0.2 micron syringe filter (VWR). All pegylatedenzyme was analyzed for lipopolysaccharide (LPS) content using a LimulusAmebocyte Lysate (LAL) kit (Cape Cod Incorporated).

Pegylated Co-hArgI was found to have nearly identical serum stability towt enzyme and displayed a k_(cat)/K_(m) value of 1690±290 s⁻¹ mM⁻¹. FIG.12 shows a denaturing gel of the final product with an apparent MW of150 kDa.

Example 15 Serum Depletion of L-Arg in the Mouse Model

Balb/c mice were treated by single IP injection with 500 μg ofpharmacologically prepared, pegylated Co-hArgI or an equal volume ofPBS. Mice were sacrificed by cardiac veni-puncture for blood collectionat the time points of 0, 48, 72, and 96 hrs. Blood samples wereimmediately mixed 50:50 (v/v) with a 400 mM sodium citrate buffer pH 4allowed to clot for 30 min and centrifuged for serum separation. Theresulting serum was then filtered on 10,000 MWCO device (Amicon) for theremoval of large proteins and precipitates and the flow-through wascollected for analysis. L-arginine standards, control mouse serum andexperiments were derivatized with OPA (Agilent) and separated on a C18reverse phase HPLC column (Agilent) (5 μm, 4.6×150 mm) essentially asdescribed by Agilent Technologies (Publication Number: 5980-3088) exceptfor modification of the separation protocol slightly by reducing theflow rate by ½ and doubling the acquisition time to get better peakseparation. An L-arginine standard curve was constructed by plottingL-Arg peak area versus concentration in order to quantify serum L-Arglevels. A single dose of pharmacologically prepared Co-hArgI wassufficient to keep L-Arg at or below detection limits for over 3 days(FIG. 10).

Example 16 HCC Tumor Xenograft Treatment with Co-Hargi

Nude mice were injected subcutaneously in the flank with ˜10⁶ HCC cellscollected from a 75% confluent tissue culture. After the HCC xenograftedtumors had grown to ˜0.5 cm³ in diameter (Day 9), mice were sorted intotwo groups. The experimental group received a 500 μg IP injection ofpharmacologically optimized Co-hArgI at day 9 and at day 12. The controlgroup received IP injections of PBS at days 9 and 12. As can be seen inFIG. 11, the PBS treated tumors had increased 3-fold in size by day 15.In stark contrast, Co-hArgI treated tumors had decreased in size by day15. Mn-hArgI treated tumors had only been shown to be retarded in growthrate (Cheng et al., 2007). Co-hArgI appears to be a highly effectivechemotherapeutic agent against HCCs both in vitro and in vivo.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is: 1-45. (canceled)
 46. A pharmaceutical compositioncomprising an isolated human Arginase I and a non-native metal cofactor,wherein the non-native metal cofactor is cobalt, the composition beingat physiological pH.
 47. The composition of claim 46, wherein the aminoacid sequence lacks an N-terminal methionine.
 48. The composition ofclaim 46, wherein the human Arginase I is covalently linked topolyethylene glycol.